Purpose :
Differences in pore formation in Schlemm’s canal (SC) endothelium between high- and non-flow areas, which may contribute to segmental aqueous outflow, have not been fully explored. Previous studies on pores used 2D scanning or transmission electron microscopy that were limited to either en face or random cross-sectional imaging, and may have missed some information. This study aims to determine the differences in pore density between high- and non-flow areas of human eyes using 3D serial block-face scanning electron microscopy (3D-EM) reconstruction.

Methods :
Two normal human eyes were perfused (15mmHg) with fluorescent tracers to label outflow. High- and non-flow areas of SC were dissected, imaged, and 3D-reconstructed. In the reconstructed areas, a total of 4225 serial SEMs (section thickness=0.13 μm) were examined by two masked observers to identify all intracellular pores (I-pores) and border pores (B-pores) and to count the total number of SC cell nuclei. Artifacts as well as focal openings with smooth edges observed in only a single section were excluded.

Results :
Pore density was higher in high- (5787.40 pores/mm2 over 0.0254 mm2, or 1.20 pores/cell; N=123 cells) vs. non-flow areas (1983.81 pores/mm2 over 0.0510 mm2, or 0.67 pores/cell; N=150 cells). Of all pores, more I-pores were observed than B-pores, in both high- (93.2% I-pores, 6.8% B-pores) and non-flow (74.3% I-pores, 25.7% B-pores) areas. Of all I-pores, the majority (69.4±17.5%) were located on the side of giant vacuoles (GVs); only 30.6±17.5% of them were located on the apical top of GVs. Interestingly, some I-pores (11.8±5.6% of all I-pores) on the side of GVs and/or near the edge of cells could have been mistaken as B-pores by 2D-SEM. Some I-pores and B-pores (12.9±7.6% of all pores) could have been missed by 2D-SEM because they were obstructed by surrounding cells/GVs.

Conclusions :
3D-EM reconstruction improved the accuracy of pore identification, as cells could be freely rotated to any viewpoint. This potentially explains the overall higher pore density we observed compared to published results (961.2 pores/mm2; Ethier et al., 1998). Also, the overall pore density is higher in high-flow areas than non-flow areas, suggesting that pore formation may play a role in the regulation of segmental outflow. Further investigation in a larger sample size using 3D-EM is warranted.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.